Oil-in-water emulsions characterization by laser granulometry and impact on γ-decalactone production in Yarrowia lipolytica

Oil-in-water emulsions composed of methyl ricinoleate (MR) or castor oil (CO) as the organic phase, stabilized by Tween 80, are in the basis of the biotechnological production of gama-decalactone. Y. lipolytica was used due to its ability to grow on hydrophobic substrates and to carry out that biotransformation. The characterization of oil droplets size distribution by laser granulometry was performed under different oil concentrations. The impact of the presence of cells on droplets size was also analyzed, as well as the relevance of washing cells. Furthermore, the granulometric characterization of the emulsions was related with gama-decalactone production for non-washed cells, the smaller droplets disappeared, using both oils, which increased gama-decalactone concentration. This suggests that the access of cells to the substrate occurs by their adhesion around larger oil droplets.The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/28039/2006) and CRUP (Cooperation project F-41/06)

Perinatal asphyxia: CNS development and deficits with delayed onset

Perinatal asphyxia constitutes a prototype of obstetric complications occurring when pulmonary oxygenation is delayed or interrupted. The primary insult relates to the duration of the period lacking oxygenation, leading to death if not re-established. Re-oxygenation leads to a secondary insult, related to a cascade of biochemical events required for restoring proper function. Perinatal asphyxia interferes with neonatal development, resulting in long-term deficits associated to mental and neurological diseases with delayed clinical onset, by mechanisms not yet clarified. In the experimental scenario, the effects observed long after perinatal asphyxia have been explained by over expression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for NAD+ during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a suitable therapeutic strategy. Asphyxia induces transcriptional activation of proinflammatory factors, in tandem with PARP-1 overactivation, and pharmacologically induced PARP-1 inhibition also down-regulates the expression of proinflammatory cytokines. Nicotinamide has been proposed as a suitable PARP-1 inhibitor. Its effect has been studied in an experimental model of global hypoxia in rats. In that model, the insult is induced by immersing rat foetuses into a water bath for various periods of time. Following asphyxia, the pups are delivered, treated, and nursed by surrogate dams, pending further experiments. Nicotinamide rapidly distributes into the brain following systemic administration, reaching steady state concentrations sufficient to inhibit PARP-1 activity for several hours, preventing several of the long-term consequences of perinatal asphyxia, supporting the idea that it constitutes a lead for exploring compounds with similar or better pharmacological profiles

An RxLR effector from phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus

The plant immune system is activated following the perception of exposed, essential and invariant microbial molecules that are recognised as non-self. A major component of plant immunity is the transcriptional induction of genes involved in a wide array of defence responses. In turn, adapted pathogens deliver effector proteins that act either inside or outside plant cells to manipulate host processes, often through their direct action on plant protein targets. To date, few effectors have been shown to directly manipulate transcriptional regulators of plant defence. Moreover, little is known generally about the modes of action of effectors from filamentous (fungal and oomycete) plant pathogens. We describe an effector, called Pi03192, from the late blight pathogen Phytophthora infestans, which interacts with a pair of host transcription factors at the endoplasmic reticulum (ER) inside plant cells. We show that these transcription factors are released from the ER to enter the nucleus, following pathogen perception, and are important in restricting disease. Pi03192 prevents the plant transcription factors from accumulating in the host nucleus, revealing a novel means of enhancing host susceptibility

Effect of promoter architecture on the cell-to-cell variability in gene expression

According to recent experimental evidence, the architecture of a promoter, defined as the number, strength and regulatory role of the operators that control the promoter, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect noise in gene expression in a systematic rather than case-by-case fashion. In this article, we make such a systematic investigation, based on a simple microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous) and how each of these affects the level of variability in transcription product from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can discriminate between different kinetic models of gene regulation.Comment: 35 pages, 6 figures, Submitte

Measurements of Elastic Moduli of Silicone Gel Substrates with a Microfluidic Device

Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Seasonality and Prevalence of Leishmania major Infection in Phlebotomus duboscqi Neveu-Lemaire from Two Neighboring Villages in Central Mali

Phlebotomus duboscqi is the principle vector of Leishmania major, the causative agent of cutaneous leishmaniasis (CL), in West Africa and is the suspected vector in Mali. Although found throughout the country the seasonality and infection prevalence of P. duboscqi has not been established in Mali. We conducted a three year study in two neighboring villages, Kemena and Sougoula, in Central Mali, an area with a leishmanin skin test positivity of up to 45%. During the first year, we evaluated the overall diversity of sand flies. Of 18,595 flies collected, 12,952 (69%) belonged to 12 species of Sergentomyia and 5,643 (31%) to two species of the genus Phlebotomus, P. duboscqi and P. rodhaini. Of those, P. duboscqi was the most abundant, representing 99% of the collected Phlebotomus species. P. duboscqi was the primary sand fly collected inside dwellings, mostly by resting site collection. The seasonality and infection prevalence of P. duboscqi was monitored over two consecutive years. P. dubsocqi were collected throughout the year. Using a quasi-Poisson model we observed a significant annual (year 1 to year 2), seasonal (monthly) and village effect (Kemena versus Sougoula) on the number of collected P. duboscqi. The significant seasonal effect of the quasi-Poisson model reflects two seasonal collection peaks in May-July and October-November. The infection status of pooled P. duboscqi females was determined by PCR. The infection prevalence of pooled females, estimated using the maximum likelihood estimate of prevalence, was 2.7% in Kemena and Sougoula. Based on the PCR product size, L. major was identified as the only species found in flies from the two villages. This was confirmed by sequence alignment of a subset of PCR products from infected flies to known Leishmania species, incriminating P. duboscqi as the vector of CL in Mali

Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

Impact of surface adhesion and sample heterogeneity on the multiscale mechanical characterisation of soft biomaterials

Funding from the Engineering and Physical Sciences Research Council (EP/L505602/1) is gratefully acknowledged